While most acute infectious diarrheas are self-limited, a subset requires aggressive treatment in order to minimize complications and/or prevent spread. Upon presentation, it is virtually impossible to know whether a diarrheal illness will have a progressive and/or fulminant course. Thus, the availability of a simple, rapid, low- cost, sensitive and specific diagnostic test would permit the delivery of directed treatment for many acute diarrheas. While testing in C. difficile associated disease is a focused first application of this work, this technology, when mature, will also find application as a bedside/field diagnostic for a wide range of infectious diarrheas. Indeed, a diagnostic that can identify a specific microbe among a panel of likely and/or potential pathogens that cause diarrhea could prove to be an important clinical-epidemiological tool worldwide that also holds promise for biodefense applications (e.g. rapid identification of clustered outbreaks of diarrheal illnesses). In this revised R21 application, significant modifications have been made to address the issues raised by the review panel, to present additional preliminary data, and to more clearly define the proposed technology as a broad-based platform suitable for a range of disease surveillance applications. The hypothesis to be tested is that microfluidic diagnostic chips can detect C. difficile in microliter-scale stool samples with a specificity and sensitivity comparable to presently available testing. The chip described here will be able to perform these tasks in a matter of minutes to hours as opposed to days. The aims of this work are to: (1) Develop a microfluidic chip-based tool to detect the presence of C. difficile bacterial DNA. The proposed work is to adapt a microfluidic technique for isolation of nucleic acids from mammalian cells and lysates to bacterial samples. Samples will be filtered and cells lysed on-chip. The isolated nucleic acids will be analyzed on-chip with real time polymerase chain reaction using primers specific for the C. difficile organism. (2) Determine whether the system can be used to detect C. difficile in human stool samples. Stool samples from individuals confirmed to be infected with C. difficile will be collected and deidentified. Samples will be processed using both on- and off-chip techniques to compare the microfluidic chip-based and gold standard benchtop methods. (3) Determine whether the microfluidic chip based diagnostic is comparable in the specificity and sensitivity to the state of the art ELISA and C. difficile cytotoxin assays. Unknown samples will be collected, analyzed and compared using the microfluidic chip and the gold standard method. Recently in the US and Canada, cases of the C. difficile associated infection has been documented in patients outside of the usual affected groups: younger people and people not in a hospital or institutional environment. This development has been a great cause of concern in the medical community, as the new strains appear to cause a more severe disease, so a diagnostic device, like the one proposed here, that can distinguish C. difficile from less serious infections quickly at the onset of symptoms will be critical for effective patient care. In addition to the use of this device in North America and Europe for emerging resistant strains of C. difficile, we envision building devices (in 6-8 years) that could distinguish between different infectious diarrheas and guide treatment in settings where antibiotic supplies are limited.